Kapitein LC et al. (2008), Microtubule cross-linking triggers the directio...

XB-ART-38336

J Cell Biol 2008 Aug 11;1823:421-8. doi: 10.1083/jcb.200801145.

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Microtubule cross-linking triggers the directional motility of kinesin-5.

Kapitein LC , Kwok BH , Weinger JS , Schmidt CF , Kapoor TM , Peterman EJ .

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Although assembly of the mitotic spindle is known to be a precisely controlled process, regulation of the key motor proteins involved remains poorly understood. In eukaryotes, homotetrameric kinesin-5 motors are required for bipolar spindle formation. Eg5, the vertebrate kinesin-5, has two modes of motion: an adenosine triphosphate (ATP)-dependent directional mode and a diffusive mode that does not require ATP hydrolysis. We use single-molecule experiments to examine how the switching between these modes is controlled. We find that Eg5 diffuses along individual microtubules without detectable directional bias at close to physiological ionic strength. Eg5's motility becomes directional when bound between two microtubules. Such activation through binding cargo, which, for Eg5, is a second microtubule, is analogous to known mechanisms for other kinesins. In the spindle, this might allow Eg5 to diffuse on single microtubules without hydrolyzing ATP until the motor is activated by binding to another microtubule. This mechanism would increase energy and filament cross-linking efficiency.

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Species referenced: Xenopus
Genes referenced: kif11 tbx2

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	Figure 1. Full-length Eg5 motility on single microtubules depends on ionic strength. (A and B) Frames (A) and kymograph (B) from a time-lapse recording showing single molecules of Eg5-GFP (green) moving directionally along a microtubule (red) in the presence of 70 mM Pipes. (C and D) Frames (C) and kymograph (D) from a time-lapse recording showing single molecules of Eg5-GFP (green) diffusing along a microtubule (red) in the presence of 70 mM Pipes plus 40 mM KCl. (E–H) MD calculated from Eg5-GFP motility in the presence of ATP (black) and ADP (red) in 70 mM Pipes plus 0, 20, 40, or 60 mM KCl. Fits represent MD = vτ. (I–M) MSD calculated from Eg5-GFP motility in the presence of ATP (black) and ADP (red) and at the indicated ionic strengths. Fits represent MD = vτ and MD = v2τ2 + 2Dτ + offset for the ATP data and MD = 2Dτ + offset for the ADP data. All numerical results are listed in Table I. (N) Histogram of the duration of binding events for 0 mM KCl and for 60 mM KCl added. Lines are single exponential fits (exp[−t/tav]) to the data (0 mM: tav = 34 ± 3, n = 212; 60 mM: tav = 16 ± 2, n = 119). Error bars represent SD. Bars, 1 μm.
	Figure 2. Dimeric Eg5 exhibits persistent microtubule association only at ionic strength well below physiological conditions. (A) Kymograph of the displacement of 230 pM Eg5-513-GFP dimers versus time in 80 mM Pipes buffer with 0.2 mM ATP. A large majority of binding events last two frames or less (<2 s). Similar results were obtained at 2 mM ATP (not depicted). (B) Kymographs showing processive runs by 14 pM Eg5-513-GFP dimers in 20 mM Pipes with 2 mM ATP. Bars, 2 μm.
	Figure 3. Full-length Eg5 motility at high ionic strength is directional on microtubule bundles (axonemes). (A) Cartoon of a possible interaction geometry of Eg5 with microtubule bundles. Note that Eg5 has a length of ∼80 nm (Kashina et al., 1996), and an axoneme has a diameter of 200 nm, with microtubule doublets being ∼70 nm apart (Alberts et al., 2002). (B) Kymographs of Eg5-GFP motility versus time on an axoneme in the presence of ATP. (C) MD calculated from motility recordings in the presence of ATP. Fit represents MD = vτ (v = 23 nm/s). (D) MSD calculated from different motility recordings in the presence of ATP (black) or ADP (red). Fit for ATP represents MD = v2τ2 + 2Dτ (v = 26 nm/s; D = 1.8 × 103 nm2/s). Fit for ADP represents MD = 2Dτ (D = 1.3 × 103 nm2/s). Error bars represent SD. Bar, 1 μm.
	Figure 4. Eg5-GFP–driven relative sliding of microtubules. (A) Cartoon illustrating assay. A sparsely labeled microtubule is surface attached, whereas another microtubule binds with an antiparallel orientation and is moved by Eg5 homotetramers. (B and C) Top kymographs show sliding of a microtubule relative to a surface-attached microtubule at 35 (B) and 28 nm/s (C). Below, corresponding kymographs of Eg5-GFP show directional runs (∼25 [B] and ∼13 nm/s [C]) between two overlapping microtubules (region marked with two red dotted lines) and diffusive motility in regions without overlap. The slope of the arrows indicate the velocity of motors (green) or microtubules (red). Bars, 2 μm.
	Figure 5. Full-length Eg5-GFP switches from diffusive to directional motility upon binding to a second microtubule. Data collected as described in Fig. 4, but in the presence of a mixture of 1 nM Eg5-GFP and 2 nM of unlabeled tetrameric Eg5. (A) Top kymograph shows sliding of a microtubule (MT) relative to a surface-attached microtubule. Below, the corresponding kymograph of Eg5-GFP shows directional runs between the overlapping microtubules (region marked with two red dotted lines). (B–F) Analysis of Eg5 motility during relative sliding. (B) Scatter plot of all pairs of short-term velocity and diffusion constant determined for a window of 15 s moving over the composite position-time trace of 94 Eg5 motors traced in the overlap zone of 11 microtubule pairs (2,335 points obtained from 2,349 s of total time). The horizontal dotted line indicates the average velocity of sliding microtubules (33 nm/s), and the vertical dotted line indicates the threshold used to discriminate slow and fast diffusion. (C and D) Similar analyses for Eg5 moving on individual microtubules at low ionic strength (C, 70 mM Pipes; Fig. 1; 4,266 points) and high ionic strength (D, data pooled from 70 mM Pipes + 60 mM KCl and 70 mM Pipes + 80 mM KCl; 2,478 points). (E) Position-time traces. Black, fraction of the composite trace used for B. Green and red, sorted time points with a short-term diffusion constant; D < 1,500 nm2/s (green) and D >1,500 nm2/s (red). (F) Histograms of the short-term velocities as obtained from the time points in the green and red trace in E. The arrow indicates the average microtubule sliding velocity. (G) Graph summarizing Eg5 behavior under various conditions. Bar, 2 μm.

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